Alan Culbertson Summary NIH F32 Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule whose presence is required for normal vascular development and endothelial integrity, though acute increases in S1P concentrations result in cardiac death. S1P binds five G protein coupled receptors (GPCRs; S1PR1-5), each of which form complexes with various G proteins (Gi, Gq, and G12/13). An array of genetic models in mice have provided a greater understanding of the S1P signaling pathways. This knowledge has led the S1P receptors to be the target of various drugs including fingolimod, the first-line treatment for relapsing multiple sclerosis. In addition, increased production of S1P leads to stimulation of S1PR1, which leads to activation of the transcription factors NF-?B and STAT3, leading to numerous types of cancer including breast cancer and lymphomas. Despite this underlying biological and medical importance, there is little in vitro analysis of these receptors and structural information is only available for S1PR1 bound to an antagonist in the inactive conformation. My objective is to determine the high-resolution structure of S1PR1 bound to Gi protein in its functional signaling complex using single-particle cryo-electron microscopy (cryo-EM). I will then characterize these interactions using mutagenesis and biophysical techniques such as microscale thermophoresis and surface plasmon resonance. In addition, I strive to obtain structural information for S1PR1-5 in its apo or ligand bound state by screening various constructs to make S1PRs larger in mass, allowing for particle alignment during cryo-EM data processing. These studies will advance the understanding of GPCR-G protein interactions and enable the engineering of novel drugs that target the S1PRs to treat numerous diseases or cancer.